US7476386B1 - Mixed lipopeptide micelles for inducing an immune response - Google Patents

Mixed lipopeptide micelles for inducing an immune response Download PDF

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US7476386B1
US7476386B1 US09/555,780 US55578098A US7476386B1 US 7476386 B1 US7476386 B1 US 7476386B1 US 55578098 A US55578098 A US 55578098A US 7476386 B1 US7476386 B1 US 7476386B1
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nef
lipopeptides
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Hélène Gras-Masse
Marc Bossus
Guy Lippens
Jean-Michel Wieruszeski
André Tartar
Jean-Gérard Guillet
Isabelle Bourgault-Villada
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Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
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    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P37/02—Immunomodulators
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to mixed lipopeptide micelles for inducing an immune response.
  • a further object is the use of these micelles for therapeutic purposes.
  • effector immune responses There are two types of effector immune responses: the humoral response due to antibodies, and the cytotoxic response due to CD8 + T lymphocytes.
  • An effective cytotoxic response requires the presentation of the antigens to the cytotoxic CD8 + T lymphocytes (CTL), in combination with class I molecules of the Major Histocompatibility Complex (MHC), but also to helper CD4 + T lymphocytes (HTL) in combination with class II MHC molecules.
  • CTL cytotoxic CD8 + T lymphocytes
  • MHC Major Histocompatibility Complex
  • HTL helper CD4 + T lymphocytes
  • lipopeptides for inducing a cytotoxic response, in other words the in vivo generation of cytotoxic T lymphocytes, has already been described.
  • application FR-90 15 870 published under the n° 2 670 787 discloses lipopeptides composed of a peptide portion comprising 10 to 40 amino acids and a lipid portion which may be derived from fatty acids or steroid groups.
  • lipopeptides show a good aptitude for inducing a cytotoxic response. However it was advisable to make them able to induce a better quality response by addition of a helper T response whose importance for effective induction and maintenance of the cytotoxic response is known. It was also advisable to make them able to induce a response in as many individuals as possible.
  • incomplete Freund's adjuvant IFA
  • the immunogenicity of the vaccine preparation used necessarily involved the functional co-presentation of the HTL and CTL units located in one or more lipopeptides in the mixture.
  • the effectiveness of the co-presentation of the different units involved depended on the combination with the incomplete Freund's adjuvant within a very fine emulsion.
  • This article also reports an experiment during which two types of associations between the HTL antigenic determinant and the CTL antigenic determinant were compared: on the one hand, a covalent sequential combination within the same lipopeptide, and on the other an association by simple mixture of a lipopeptide containing the CTL unit with a peptide containing the HTL unit.
  • the results of this study showed a very clear advantage of the covalent combination compared to the mixture, as performed by the authors, in other words by mixture of solutions containing DMSO and PBS buffer (the peptides or lipopeptides were kept in stock solutions at a concentration of 10-20 mg/ml and diluted with PBS just before use).
  • this article mentions the combination of a lipopeptide and a peptide, and not of two lipopeptides. For this reason no mixed micelle formation could take place.
  • This article thus does not describe a mixture of lipopeptides independently containing a CTL antigenic determinant and an HTL antigenic determinant, but the mixture of a lipopeptide containing an HTL antigenic determinant and a nonapeptide corresponding to a minimal CTL antigenic determinant.
  • the formation of mixed micelles or of micro-aggregates there is no mention of the formation of mixed micelles or of micro-aggregates.
  • the possibility of direct combination between the nonapeptide and the class I MHC expressed at the surface of the cells could explain the success of the approach followed.
  • the immunogenicity of the preparation indicates that there was effectively co-presentation of the HTL and CTL antigenic determinants by the same antigen-presenting cell; however, the minimal nonapeptide used has the capacity to link directly with the class I MHC at the surface of the antigen-presenting cell, without its presentation by the cell being necessary.
  • compositions containing within the same micelles, or the same micro-aggregates, on the one hand lipopeptides presenting a CTL antigenic determinant and on the other lipopeptides containing an auxiliary T antigenic determinant, i.e. mixed micelles or micro-aggregates, have never, to the knowledge of the applicant, been described.
  • the object of the present invention is thus micelles or micro-aggregates for inducing an immune response containing at least:
  • FIG. 1 represents the chemical structure of the resin of type KNORR-MBHA.
  • FIGS. 2 and 3 show the two-dimensional nuclear magnetic resonance (2DNMR) spectra of a single lipopeptide (lipopeptide ENV) and a mixture of lipopeptides respectively.
  • FIG. 4 shows the helper response of eight macaques immunized with a mixture of lipopeptides.
  • FIGS. 5A to 5F show the cytotoxic response of macaque n° 109.
  • FIGS. 6A to 6D show the cytotoxic response of macaque n° 129.
  • FIGS. 7A and 7B show the cytotoxic response of macaque n° 127.
  • FIGS. 8 , 9 , 10 and 11 respectively show the cytotoxic responses of macaques n° 102, 105, 120 and 125.
  • FIGS. 12A , 12 B and 12 C respectively show the anti-N1, anti-G2 and anti-E cytotoxic activities of PBMC, CD8 + and CD4 + cells of the individual V4.1.
  • FIG. 13 shows the cytolytic activity of PBMC of individual V4.5 collected twenty weeks after the beginning of immunization, stimulated in vitro with peptide N2 then tested for their CTL activity against wild vaccine (WT), or this same virus expressing a recombinant NEF protein (NEF, NEF-2, NEF-MN, NEF-A, NEF-ROD).
  • WT wild vaccine
  • NEF-MN a recombinant NEF protein
  • immune response means the whole of the induced immune response, which includes the cytotoxic response and the humoral response.
  • the micelles according to the present invention are not limited to two lipopeptides, but may contain other lipopeptides independently presenting HTL or CTL antigenic determinants.
  • helper T antigenic determinant should be understood as meaning an amino acid sequence able to bind with at least one class II HLA receptor, and able to be recognised by helper T lymphocytes.
  • CTL antigenic determinant should be understood as meaning an amino acid sequence able to bind with at least one class I HLA receptor and able to be recognised by cytotoxic T lymphocytes.
  • multivalent helper antigenic determinants The helper T antigenic determinants able to bind with several different class II HLA receptors are called multivalent helper antigenic determinants (multivalent HTL).
  • micelles or micro-aggregates should be understood aggregates of lipopeptides with a size making them able to be assimilated simultaneously by any antigen-presenting cell (APC) and preferably with a size less than about 1 ⁇ m.
  • APC antigen-presenting cell
  • the mixed micelles according to the invention in other words comprising lipopeptides containing cytotoxic antigenic determinants and lipopeptides containing helper T antigenic determinants, have the advantage of combining, within the same microvolume which can be assimilated by a single APC, a wide variety of CTL and HTL antigenic determinants, without their covalent combination being necessary, while respecting the required criterion of chemical definition.
  • Micelles which each contain a single type of lipopeptide, containing a CTL antigenic determinant or an HTL antigenic determinant do not result in an effective co-presentation corresponding to the induction of a strong effector response.
  • obtaining a CTL response by the use of mixed micro-aggregates or micelles avoids the use of emulsions with oily adjuvants, such as incomplete Freund's adjuvant, whose use is not approved in human therapeutics.
  • the micelles and micro-aggregates according to the present invention are however compatible with the use of emulsions with clinically acceptable oily vehicles.
  • a further advantage of the mixed micro-aggregates or micelles according to the present invention, each containing at least two types of lipopeptides, is that the solubilization of lipopeptides with a low solubility in water or in clinically acceptable solvents, or insoluble lipopeptides, may be improved by their combination with other lipopeptide(s) with better solubility.
  • the micelles according to the present invention also show the advantage, compared to lipopeptides in which the HTL and CTL units are combined covalently and whose size is limited, such as those described by VITIELLO et al. (1995, cited above), of allowing the combination of a wide variety of units, and thus can be used for the vaccination of human or animal populations not selected on the basis of genetic restriction.
  • the micelles according to the present invention may contain a lipopeptide with at least a CTL antigenic determinant and another lipopeptide containing at least a helper antigenic determinant.
  • such micelles may also contain several different lipopeptides containing different cytotoxic antigenic determinants and different lipopeptides with different helper antigenic determinants.
  • the lipid units of the lipopeptides may independently be one or more C 4 -C 18 units, and in particular one or more C 4 -C 18 chains derived from fatty acids, or fatty alcohols, optionally branched and unsaturated or derived from a steroid.
  • They may contain one or two C 4 to C 18 lipid chains linked by a covalent bond to one or two amino acids of the peptide part. They may also be composed of two palmitic acid chains linked to the alpha and epsilon NH 2 groups of a lysine.
  • lipid units may also be composed of, or contain, a residue of palmitic acid, 2-aminohexadecanoic acid, oleic acid, linoleic acid, linolenic acid, pimelautide, trimexautide, or a derivative of cholesterol, or any other natural lipid component of the cell membranes.
  • the lipopeptides constituting the mixed micelles or micro-aggregates are advantageously water-soluble in a proportion of at least 30% (by weight). These water-soluble lipopeptides have cationic surface-active properties, suitable for providing a solubilizing effect on other lipopeptides in weak acid medium.
  • the non-lipid part contains between 10 and 100, and preferably between 10 and 50 amino acids.
  • the number of amino acids depends on the number of antigenic determinants constituting the non-lipid part of the lipopeptide and on their sizes, on the nature of the lipid part, and the proportions of the lipid and non-lipid parts.
  • HTL and CTL antigenic determinants used are advantageously antigenic determinants able to bind with several different HLA, otherwise called multivalent or promiscuous antigenic determinants.
  • the HTL antigenic determinant used is preferably composed of the multivalent peptide 830-843 of the tetanus toxin.
  • the glutamine (Q) of this sequence may optionally be acetylated.
  • multivalent HTL antigenic determinants may be the multivalent antigenic determinant of hemagglutinin (PREVOST-BLONDEL et al., 1995, J. Virol., vol. 62, n° 12, pages 8046-8055) or the PADRE antigenic determinant (ALEXANDER et al., 1994, Immunity, 1, 751).
  • the CTL antigenic determinant may be any antigenic determinant able to activate cytotoxic CD8 + T lymphocytes.
  • a protein presented by a tumour cell and in particular by a melanoma a protein from HIV, from hepatitis B virus (HBV) or from papillomavirus, or protein p53.
  • the antigenic determinants of protein p53 may in addition be comprised in the sequences 25-35, 63-73, 129-156, 149-156, 187-205, 187-234, 226-264, or 249-264 of this protein.
  • CTL antigenic determinants with a capacity to bind to class I HLA may be those included in the peptide 43-57 of HPV (GQAEPDRAHNIVTF (SEQ ID NO:284)) which contains HLA A2, A24, B7 and B18 antigenic determinants.
  • the CTL antigenic determinants may also be those of parasite antigens, and in particular of Plasmodium falciparum.
  • the mixed lipopeptide micro-aggregates or micelles according to the present invention may be freeze-dried, then taken up into any clinically acceptable buffer to be administered to the patients to be treated, and in particular to patients to be vaccinated.
  • They may be administered by any administration route used in therapeutics and, as non-limiting examples, by parenteral, percutaneous, oral, or sublingual routes or by intra-pulmonary nebulizer.
  • a further object of the present invention is thus the use of these lipopeptides for the production of a drug or vaccine for inducing a specific immune response, and in particular, for inducing an immune response against cancers such as melanoma, HIV and HBV viruses, papillomavirus, p53 or malaria.
  • Another object of the present invention is a pharmaceutical composition characterized in that it contains a pharmacologically active quantity of one or more of the lipopeptides described above, in addition to pharmaceutically compatible vehicles.
  • the present invention also relates to a method of inducing an immune response against an antigen comprising the administration of micelles or micro-aggregates, such as those described above, to an individual for which such a response is sought.
  • An additional object is a method of immunization against a pathogenic agent comprising the administration of micelles or micro-aggregates such as those described above to an individual for whom such an immunization is sought.
  • pathogenic agents, and antigens may be those listed above.
  • the lipopeptides forming the micelles according to the present invention may be produced by any suitable method known to a person skilled in the art. They may in particular be obtained by the Boc-benzyl or Fmoc-tert-butyl methods, in particular as disclosed in the application FR-90 15 870, which patent application is incorporated herein by reference.
  • the introduction of the lipid chain may be achieved in the solid phase, after selective deprotection of the functional group or groups concerned, as described in the article by DEPREZ et al., (1996, Vaccine, volume 14, n° 5, 375-382).
  • the lipid chain may be introduced onto the ⁇ -NH 2 function of a lysine protected on the ⁇ -NH 2 function by an F-moc group.
  • the Fmoc-lys (Palm) obtained may then be used in solid-phase synthesis to produce the lipopeptide.
  • the micelles and micro-aggregates according to the present invention may be obtained by dispersing each lipopeptide in a concentrated acetic acid solution at about 80% concentration, then mixing the solutions thus obtained.
  • the quality of dissolution i.e. the effective dispersion at the molecular level of each lipopeptide before the preparation of the mixture, is confirmed by the two-dimensional nuclear magnetic resonance method (2DNMR).
  • 2DNMR two-dimensional nuclear magnetic resonance method
  • the resolution of the signal obtained during homonuclear experiments in two dimensions in a 600 MHz field confirms the complete dispersion, at the molecular level, of the lipopeptides in solution.
  • the clarity of the mixture is not a sufficient criterion: in particular, the taking up of the lipopeptides by DMSO or a DMSO/water mixture does not lead, in most cases, to a sufficient dispersion state, which explains the ineffectiveness of the mixture studied by VITIELLO et al. (1995, cited above).
  • Dissolution by acetic acid/water mixtures which are more dilute in acetic acid also does not lead in all cases to the preparation of a mixture of mixed micro-aggregates or micelles containing a statistical proportion of each constituent of the mixture at the microvolume level.
  • the sterilizing filtration over a 0.22 ⁇ m membrane is either impossible, or irregular, with filtration yields which differ according to the constituents, which indicates that at the scale of a particle of this size, the representation of each constituent of the mixture has not been achieved.
  • This micro-heterogeneity compromises the immunogenicity of the mixture, since it comprises the simultaneous capture and presentation of all constituents by a single antigen-presenting cell (APC), in the case of CTL and HTL antigenic determinants present on separate lipopeptides.
  • APC antigen-presenting cell
  • RPNNNTRKSI HLA-B27 PPIPVGEIY SEQ ID NO. 9 HLA-B35 KRWIILGLNK SEQ ID NO. 10 HLA-B27 LGLNKIVRMY SEQ ID NO. 11 HLA-B62 QVPLRPMTYK SEQ ID NO. 12, 168, 174, 215 HLA-A-3, A11, B27.2 VPLRPMTY SEQ ID NO. 13 HLA-B35 AVDLSHFL SEQ ID NO. 14 HLA-B62 AVDLSHFLK SEQ ID NO. 15, 175 HLA-A11 TQGYFPDWQNY SEQ ID NO. 16 HLA-B62 YFPDWQNYT SEQ ID NO.
  • HLA-B17 17 HLA-B17, B35 TPGPGVRYPL SEQ ID NO. 18, 193 HLA-B7 RYPLTFGW SEQ ID NO. 19 HLA-B27.2 YPLTFGWC SEQ ID NO. 20 HLA-B18 AFHHVAREL SEQ ID NO. 21 HLA-B52 FLKEKGGL SEQ ID NO. 22, 200 HLA-B8
  • the solid phase approach was selected, using the Fmoc strategy for protecting the ⁇ -amine function, and t-Bu for protecting the side chains.
  • the protocol used was a standard protocol based on the synthetic methods described by ATHERTON (Solid-phase synthesis, a practical approach, IRL Press, 1989) and FIELDS and NOBLE (Int. J. Pept. Prot. Res., 1990, 35, 161-214).
  • the Fmoc-Lys(Palm)-OH was coupled to a resin of KNORR-MBHA type ( FIG. 1 ). After deprotection of the alpha-amine function, the first amino acid was coupled (for example Fmoc-Leu-OH in the case of NEF 66).
  • the coupling agent was TBTU (3 eq) in the presence of DIPEA (4.5 eq), with verification of coupling by a calorimetric test. A systematic acetylation was performed after a negative reaction had been obtained with this test, to minimize the risk of obtaining peptides by deletion. This succession of operations was repeated until all the amino acids in the sequence had been added.
  • the peptides were deprotected and cleaved by a TFA/water/DTT mixture (NEF 66, ENV), TFA/water/DTT/Ac-Trp-OH (GAG 183, GAG 253, NEF 117) or TFA/water/EDT/Ac-Trp-OH (NEF 182).
  • the peptides were each purified on a Vydac C18 column which was exclusively used for this purpose, at ambient temperature, with a water-acetonitrile solvent system, in perchlorate or TFA buffer.
  • Each peptide was produced from a single batch of synthesis and purification. No recycling of purification fractions was performed.
  • the peptides NEF 66, NEF 117, NEF 182 and ENV could be dissolved in pure water, at concentrations of up to 5 mg/ml. Peptide NEF 117 however gave a slightly opalescent solution. Peptides GAG 182 and GAG 253 were not soluble under these conditions.
  • the mixture of lipopeptides was however soluble in pure water, indicating that the hydrophilic lipopeptides were having a solubilizing effect on the less soluble peptides.
  • DMSO dimethyl sulfoxide
  • 80% concentrated acetic acid was then tested.
  • the peptides were dissolved at a concentration of 1 mM in 1 ml of 80% acetic acid (corresponding to: NEF 66: 3.86 mg/ml; NEF 117: 4.02 mg/ml; NEF 182: 3.45 mg/ml; GAG 183: 3.98 mg/ml; GAG 253: 4.063 mg/ml; ENV: 4.027 mg/ml).
  • the lipopeptide samples were prepared by dissolving the lipopeptides in a solution of acetic acid (CD3COOD, 99.5% D atoms, EURISO-TOP, France)/H2O; 80:20 (V:V). 4 ⁇ l of a 50 mM solution of TMSP [sodium 3-(trimethylsilyl)propanesulfonate] in D2O were added as chemical shift reference. The final concentration of each peptide was 1 mM in at least 2 ml of solvent, which were transferred into 8 mm diameter NMR tubes (WILMAD 513A-7PP, Interchim, France).
  • the proton NMR spectra were performed on a BRUKER DMX600 NMR spectrometer fitted with an 8 mm BBI probe with z gradient, at a sample temperature of 310° K.
  • NOESY Nuclear-Overhauser effect spectroscopy experiments in two homonuclear dimensions according to Kumar et al. (1980, Biochem. Biophys. Res. Comm., 95, 1-6) and TOCSY (Total Correlation Spectroscopy) according to Bax and Davis (1985, J. Magn. Reson., 65, 355-360) and Griesinger et al. (1988, J. A. C. S., 110, 7870-7872) were obtained with 2048 ⁇ 512 complex points and processed after multiplication in two dimensions by a sine wave displaced by ⁇ /4 with 2048 ⁇ 1024 points, for a spectral window of 12 ppm.
  • the mixture times were 300 ms for the NOESY and 160 ms for the TOCSY.
  • a MLEV 16 was applied with a B1 field of 7.8 KHz. So as to be under the same temperature conditions, the spin-lock time of the TOCSY was applied without resonance (+ or ⁇ 1 MHz) in the NOESY. The suppression of water was achieved by using a slight pre-saturation of this signal during the relaxation time and the mixture time of the NOESY.
  • the 2D NMR spectrum of peptide ENV is shown on FIG. 2 .
  • the spectra of all the peptides could be obtained under the same conditions and interpreted.
  • a 2D NMR spectrum of a virtual mixture was obtained by superimposing the 6 spectra obtained individually onto a single representation. It was compared with the 2D NMR spectrum actually obtained by mixing the solutions ( FIG. 3 ). The resolution of the signals remained comparable, proving that none of the peptides had altered the solubility of the other constituents of the mixture.
  • the sequence analysis required an accumulation of signals over 120 hours for each lipopeptide, during which period no significant alteration of the peptides was detected, either by NMR or by RP-HPLC. This observation thus allows the use of this solvent for the solubilization of the lipopeptides, their mixture, then filtration, even with a residence time of the order of 1 to 2 hours, conceivably necessary for the handling of relatively large volumes.
  • Tests on the sterilizing filtration were performed on 5 mg/ml solutions of each lipopeptide in water for peptides NEF 66, NEF 117, NEF 182 and ENV and in 25% acetic acid for peptides GAG 183 and GAG 253.
  • the filtration yields for 1 ml over Millipore Millex GV SLGV 0130S filters (0.22 ⁇ m), followed by freeze-drying, are shown in table 8 (to within the precision of the determination).
  • the solutions were mixed, then filtered over Millex GV SLGV 0130S filters (0.22 ⁇ m). The filtration required a lower pressure than during the filtration of the 8% solution.
  • the receiver vessels and the filter were then rinsed with water, in sufficient quantity to give a final acetic acid concentration of 8% (final volume 15 ml as before), so as to ensure the quality of the freeze-drying step.
  • the filtration yields of the peptides in the mixture were calculated for each lipopeptide, to give the results listed in the final column of table 10 (to within the precision of the determination).
  • the homogeneity of the yields confirms the homogeneity of the solution resulting from the dispersion at the molecular level at the time of filtration in concentrated acetic acid.
  • the subsequent dilution cannot result in a reorganization of each peptide into monovalent entities, by application of the laws of entropy.
  • This method of preparation of the mixture thus gives mixed micelles which each necessarily contain a statistical representation of each lipopeptide.
  • the surface-active properties of the lipopeptides can operate and guarantee the solubility in water of vaccine doses after freeze-drying as well as the stability of the solutions during the handling time.
  • the procedure used was the same as for the previous batch, apart from the quantities.
  • the solution of the peptide (20 mg/ml in 80% acetic acid) was filtered in 4 portions, changing the filter before its saturation, using identical membranes (Durapore STERIVEX GV 0.22 ⁇ m sterile units (Millipore)), then made up with the water used for rinsing the filters and for dilution.
  • the final volume was 1516 ml (including 154 ml of acetic acid: 10% in final solution).
  • the portion volume was 1.3 ml per dose.
  • the apportioned doses were freeze-dried, and analysed using a validated HPLC determination method.
  • the filtration yield for each lipopeptide is given below in table 11, and takes into account the determination sensitivity of each lipopeptide.
  • the mixture after dilution and freeze-drying gave a white powder forming a compact homogeneous cake, which could very easily be taken up into solution in pure water or a solvent able to restore the osmolarity of the solution (5% glucose, 5% mannitol).
  • the solution showed a very slight opalescence.
  • the pH obtained after taking up in a non-buffered solvent was 4.90. Raising the pH by 1 unit caused a slow precipitation: this behaviour contributed to the formation of a deposit during subcutaneous or intramuscular injection.
  • powders for parenteral use are subjected to a requirement of uniformity of concentration.
  • the test must be performed on 10 random samples, which are analysed individually for the active ingredient using an appropriate analytical method.
  • the preparation satisfies the test if the concentration of each sample is between 85 and 115% of the average concentration.
  • the test was performed on 15 random samples, taken up in solution and diluted in 80% acetic acid according to a standardized operational procedure, so as always to inject an identical proportion of about 15 ⁇ g, defined during preparation of the calibration curve. Each sample was injected three times, the concentration of each active ingredient corresponding to the average of the three values obtained.
  • This small batch was prepared in order to perform a pre-clinical test on macaques, to verify the tolerance and immunogenicity. This batch resulted from the mixture of the following lipopeptides:
  • the macaques were immunized by subcutaneous injection of the batch prepared above (500 ⁇ g), in sterile water, and were reinjected after periods of thirty days and sixty days.
  • PBMC Blood cells
  • PBMC Blood cells
  • Anti-peptide CTL lines were obtained by cultivating the monkey PBMC (2 ⁇ 10 6 cells/ml) in microtitration plates, in RPMI 1640 supplemented with penicillin (100 U/ml), streptomycin (100 ⁇ g/ml), L-glutamine (2 mM), non-essential amino acids (1%), sodium pyruvate (1 mM), HEPES buffer (10 mM), 2-mercaptoethanol (2 ⁇ 10 ⁇ 5 M) and 10% fetal calf serum (FCS) inactivated by heat.
  • RPMI 1640 supplemented with penicillin (100 U/ml), streptomycin (100 ⁇ g/ml), L-glutamine (2 mM), non-essential amino acids (1%), sodium pyruvate (1 mM), HEPES buffer (10 mM), 2-mercaptoethanol (2 ⁇ 10 ⁇ 5 M) and 10% fetal calf serum (FCS) inactivated by heat.
  • the effector cells were stimulated by addition of new autologous PBMC, which had been in contact with the peptide mixture (5 ⁇ M of each) for two hours, then washed and irradiated (4000 rads).
  • PBMC cells (2 ⁇ 10 5 in 200 ⁇ l per well) were cultivated in plates containing 1 ⁇ g/ml of lipopeptide TT (830-846), and 10 ⁇ g/ml of the peptide from tetanus toxin (TT).
  • the phenotype of the cell lines was determined the day that the chromium release assay was performed, by incubating the cells with anti-CD4 conjugated to FITC (OKT4, Ortho Diagnostic Systems, Raritan, N.J.) and with anti-CD8 conjugated to phycoerythrin (Leu-2a, Becton Dickinson, Mountain View, Calif.) for thirty minutes at 4° C.
  • the cells were washed with PBS buffer, then the percentage of coloured cells was determined using an Epics Elite flow cytometer (Coulter, Margency, France). Antibodies presenting a mixture of isotypes were used as controls.
  • B (B-LCL) cell lines were obtained by incubating series dilutions of PBMC using the supernatant of cell line S 594. This line is infected by baboon herpes virus which immortalizes the cells (herpes virus papio). The B-LCL were then cultivated in the culture medium supplemented with 10% FCS.
  • the target cells were sensitized with the peptides.
  • 10 6 B-LCL cells were incubated either overnight or for 1 hour, respectively, with the long or short peptides (concentration range 10 ⁇ 5 M-10 ⁇ 8 M) at 37° C. in a humid atmosphere with 5% CO 2 .
  • the B-LCL were incubated at a concentration of 10 6 cells/ml with a recombinant vaccine virus (20 PFU/cell) for eighteen hours under the same conditions.
  • the B-LCL were then washed and marked with 100 ⁇ Ci Na 2 51 CrO 4 (NEN Life Science Products, Courtaboeuf Les Ullis, France) for 1 hour, washed twice and used as target cells.
  • the 51 Cr release was performed in microtitration plates.
  • the cytolytic activity of the anti-SIV cell lines was measured by mixing 5 ⁇ 10 3 target cells marked with chromium with the effector cells, at various ratios of effector cells to target cells, in a final volume of 200 ⁇ l/well. The plates were incubated for 4 hours at 37° C., then 100 ⁇ l of supernatant was taken from each well and analysed in a gamma radiation counter.
  • the spontaneous release of chromium was determined by incubating the target cells with medium alone. It never exceeded 20% of the total chromium incorporated.
  • the specific release of chromium was measured as follows: 100 ⁇ (experimental cpm ⁇ spontaneous cpm )/(maximum cpm ⁇ spontaneous cpm ).
  • FIG. 4 shows the helper T response of the eight macaques.
  • FIGS. 5 to 11 show the cytotoxic response of the macaques.
  • the effectiveness of the induction of a CTL response confirms that the APC of the animals were able to capture and present one or more CTL antigenic determinants, and simultaneously the strong helper antigenic determinant present in the tetanus anatoxin and recognized by some of the animals.
  • a mixture of lipopeptides was defined for performing a clinical test (VAC 10), combining within the micelles the same peptide TT with sequences selected on the selection principle developed for VAC 04 (existence of one or more CTL antigenic determinants per sequence).
  • composition of this mixture in which Pam represents a unit derived from palmitic acid and Ac the acetyl group, was the following:
  • This set of peptides was synthesized as described in the previous examples.
  • the mixture of solutions was performed on a sample of 5 mg of each peptide, dissolved at a concentration of 20 mg/ml in 80% acetic acid then mixed in the following order: 1: GAG 17; 2: NEF 66; 3: NEF 116; 4: TT; 5: GAG 253; 6: POL 325.
  • This set of peptides was synthesized as described in example 1.
  • the mixture of solutions was performed on a sample of 5 mg of each peptide previously dissolved at a concentration of 20 mg/ml in 80% acetic acid then mixed in the following order: 1: LSA3 NRI; 2: LSA3 NRII; 3: LSA3 CT1; 4 LSA3 RE.
  • the micelles which were injected were obtained as described in example 1 for batch CK9.
  • N1 N1 (NEF 66 to 97), N2 (NEF 117 to 147), N3 (NEF 182 to 205), G1 (GAG 183 to 214), G2 (GAG 253 to 284) and E (ENV 303 to 335).
  • NEF 73-82, NEF 84-92 and EBNA 4 (SEQ ID NO:225) 416-424HLA restricted to HLA-A11.
  • NEF 134-141 and GAG 263-272 (SEQ ID NO:10) restricted to HLA-B27.
  • Volunteers V4.15, 4.16, V4.17, V4.18, and V4.28 were immunized with 500 ⁇ g of each of the six lipopeptides.
  • Volunteer V4.5 received 100 ⁇ g of the 6 lipopeptides, while volunteers V4.1, V4.19, V4.21, V4.32 and V4.34 each received 500 ⁇ g of the six lipopeptides.
  • PBMC Peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • PDA phytohemagglutinin A
  • TT tetanus toxin
  • SEB enterotoxin B of Staphylococcus golden, Reference Sigma S4881
  • In vitro stimulation of the PBMC was performed by mixing 106 PBMC (responsive cells) with 106 irradiated stimulant cells (autologous PBMC incubated for 2 hours with different peptides) in complete RPMI culture medium (RPMI 1640 supplemented with 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM Hepes, nonessential amino acids and 10% heat-inactivated bovine serum albumin).
  • RPMI 1640 supplemented with 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM Hepes, nonessential amino acids and 10% heat-inactivated bovine serum albumin.
  • interleukin-2 10 U/ml of interleukin-2 were added after 3 days.
  • the responsive cells were restimulated each week for 3 or 4 weeks using peptides incubated with autologous PBMC (prepared in the same way as on day 0), in a medium supplemented with 10 U/ml of interleukin-2.
  • the CTL cells were tested using the EBV autologous cell line as target overnight with 10 ⁇ g of the different peptides (N1, N2, N3, G1, G2 or E) for 10 6 cells.
  • EBV target cells were infected, at a rate of 10 6 cells/ml, with a wild type (WT) vaccine virus or with HIV-1/LAI, HIV-1/MN, HIV/A or HIV/ROD NEF recombinant vaccine viruses overnight (20 PFU/cell).
  • WT wild type vaccine virus
  • HIV-1/LAI HIV-1/MN
  • HIV/A HIV/ROD NEF recombinant vaccine viruses
  • the different target cells were then washed and marked with 100 ⁇ Ci of Na 2 51 CrO 4 (NEN Life Science Products, Les Ullis, France).
  • the cytolytic activity was measured in a 51 Cr release test, over 4 hours. The average spontaneous release did not exceed 20% of the total 51 Cr incorporation.
  • 96-Well microcells plates (MultiScreen-HA, Millipore S.A., Molsheim, France) were covered with 5 ⁇ g/ml of mouse anti-human- ⁇ -interferon antibody, as capture antibody (Genzyme Corporation, Cambridge, Mass., USA) overnight at 4° C.
  • alkaline phosphatase chromogenic substrate Bio Rad Laboratories, Hercules, Calif., USA
  • the negative control consisted of PBMC incubated alone in the medium, or incubated with a peptide corresponding to a CD8 + antigenic determinant derived from the HIV virus presented by adapted HLA.
  • the positive control consisted of activating the PBMC with 50 mg/ml of PMA (Phorbol myristate acetate, reference Sigma P 8139) and 500 ng/ml of ionomycin (100 to 300 PBMC per well were added).
  • PMA Phorbol myristate acetate, reference Sigma P 8139
  • ionomycin 100 to 300 PBMC per well were added.
  • This strong mitogen stimulation allowed measurement of the viability of the T lymphocytes, and verification of the quality of the storage in the cold.
  • the serums from the immunized volunteers were tested by ELISA for the presence of IgG antibody directed against the NEF (N1, N2, N3), GAG (G1, G2), and ENV (E) peptides
  • anti-N1 IgG antibodies were detected in five of the vaccinated subjects (V4.6, V4.28, V4.1 (SQ21), V4.32 (QS21), and V4.34 (QS21)), and anti-N2 IgG antibodies were detected in the serums of ten of the subjects, among the twelve vaccinated. No antibody of type anti-N3 IgG was detected.
  • the titration in anti-N2 antibody was negative in the serums of individuals V4.17 and V4.18. The antibody titration was three to five times 1 greater than that of the negative control in the serums of V4.15, V4.16, V4.1 (QS21), V4.5 (QS21) and V4.21 (QS21).
  • the antibody titration was five to ten times greater than that of the negative control in the serums of V4.6, V4.19 (QS21), and V4.28. Finally, the serums of patients V4.32 (QS21) and V4.34 (QS21) showed antibody titration at least 10 times greater than that of the negative control.
  • the anti-G2 antibody titration was 2 to 3 times greater than that of the negative control for patient V4.18 (QS21), the antibody titration was 5 to 10 times greater than that of the negative control for individuals V4.16, V4.17, V4.5 (QS21), V4.19 (QS21) and V4.21 (QS21).
  • the serums of patients V4.6, V4.15, V4.28, V4.1 (QS21), V4.32 (QS21) and V4.34 (QS21) had an antibody titration more than ten times greater than that of the negative control.
  • the serums of 6 of the 12 individuals tested, V4.28, V4.1 (QS21), V4.5 (QS21), V4.19 (QS21), V4.32 (QS21) and V4.34 (QS21) contained specific anti-E antibodies.
  • the NEF, GAG and ENV peptides caused proliferation of the donor PBMC only, after the vaccination.
  • the PBMC of the individuals immunized with the lipopeptides (with or without QS21 adjuvant) proliferated against at least one peptide after 20 weeks (4 weeks after the third injection, for 8 subjects out of 10 given in table 15).
  • the PBMC of individual V4.6 proliferated in response to peptides N3, G1 and G2 with a proliferation index of between 4 and 10.
  • An induction of the proliferation in response to G1, G2 and E was observed with the PBMC of individual V4.16.
  • the PBMC of individual V4.28 were able to proliferate in response to peptides N1, N3, G2 and E.
  • a proliferative response against peptides N1, G2 and E was observed for the PBMC of vaccinated individual V4.5.
  • the removal experiments performed with the PBMC from the different individuals vaccinated showed that the proliferation of the PBMC recovered after twenty weeks occurred preferentially via the helper CD4 + T cells.
  • the PBMC obtained before and after the immunizations were stimulated in vitro and tested for their CTL activity specific to HIV.
  • the specific CTL activity was tested against the EBV autologous cell line, incubated with or without the NEF, GAG and ENV peptides. No anti-HIV response was detected with the PBMC recovered before the immunization. A specific CTL activity was detected in the PBMC collected three weeks after the immunization for nine of the twelve individuals.
  • Table 16 summarizes the cytotoxic activity of eight of the vaccinated individuals, the activity of one other individual being shown in FIG. 12 .
  • At least one peptide contained in the lipopeptide vaccine induced specific CTL effector cells recognizing the HIV peptides.
  • the PBMC of individual V4.6 recognized in a cytotoxic test the EBV autologous cells stimulated with peptides G2 and E.
  • the PBMC of individual V4.16 recognized peptide N3 and E.
  • the percentage of lysis was variable, weak for individual V4.16 recognizing peptide N3, intermediate for individual V4.18 with peptide N1 and strong for individual V4.5 (QS21) with peptides N2 and G2.
  • a specific CTL activity was also generated against peptides containing a minimal CD8 + HIV antigenic determinant (individuals V4.16 and V4.28).
  • the CD8 + or CD4 + T lymphocytes were removed from the PBC and a cytotoxicity test was performed.
  • CTL cytotoxic T cells
  • PBMC from individual V4.5 collected 20 weeks after immunization, and stimulated in vitro with peptide N2, were thus tested for their CTL activity against autologous targets infected with different viruses expressing the recombinant NEF protein.
  • the results of representative experiments are given in FIG. 13 .
  • Anti-peptide N2 CTL obtained from individual V4.5 (QS21) recognized an antigen naturally modified by autologous EBV-LCL infected with recombinant viruses of the vaccine coding for the HIV-NEF genes obtained from different strains of HIV.
  • CTL specific to the HIV virus recognized NEF-LAI and NEF-MN with the same effectiveness.
  • a lower percentage of specific lysis was obtained for the NEF-A protein or the NEF-ROD protein.
  • Effector CD8 + T cells may exercise a lytic activity and/or produce lymphokines.
  • the quantity of CD8 + T cells secreting ⁇ -interferon was thus evaluated by a specific ELISPOT test.
  • An ELISPOT has also been used to quantify ex vivo the number of CD8 + T cells secreting ⁇ -interferon specific to HIV peptides in the PBMC of the vaccinated subjects (table 17).
  • HLA-A2 MLLAVLYCL 1-9 249 HLA-A2 YMNGTMSQV 369-377 250 YMDGTMSQV 252 HLA-A24 AFLPWHRLF 206-214 251 HLA-B44 SEIWRDIDF 192-200 253 HLA-DR4 QNILLSNAPLGPQFP 56-70 254 SYLQDSDPDSFQD 450-462 255 Pmel17 gp100 HLA-A2 KTWGQYWQV 154-162 256 HLA-A2 AMLGTHTMEV 177-186 257 HLA-A2 MLGTHTMEV 178-186 258 HLA-A2 ITDQVPFSV 209-217 259 HLA-A2 YLEPGPVTA 280-288 260 HLA-A2 LLDGTATLRL 457-466 261 HLA
  • Tumour Antigenic determinants resulting from de mutations Amino SEQ Gene/ MHC acid ID protein Tumour restriction Peptide positions NO: MUM-1 Melanoma HLA-B44 EEKLIVVLF 30-38 270 CDK4 Melanoma HLA-A2 ACDPHSGHFV 23-32 271 ⁇ -catenine Melanoma HLA-A24 SYLDSGIIF 29-37 272 HLA-A2 Renal car- — cinoma CASP-8 Squamous HLA-B35 FPSDSWCYF 476-484 273 carcinoma of head and neck
  • the three injections of the six lipopeptides were administered at 0.4 and 16 weeks.
  • the detection of the specific antibodies of the peptides of the HIV virus was performed using an ELISA assay with serum dilution to 1/100.
  • the ELISA assay plates were covered with NEF 66-97 (N1), NEF 117-147 (N2), NEF 182-205 (N3), GAG 183-214 (G1), GAG 253-284 (G2) or V3 ENV 303-335 (E).
  • the PBMC of the volunteers were recovered before injection of the lipopeptides (W0), and during the twentieth week (W20).
  • c 2 10 5 cells were cultivated with 1 ⁇ g/ml of the HIV lipopeptides and the proliferation was measured by incorporation of tritiated thymidine at day 6.
  • the lipopeptides were N1, N2, N3, G1, G2, and E.
  • the proliferation index obtained with the culture medium only was equal to 1.
  • the proliferative response (cpm) of the PBMC of the volunteers cultivated in the medium alone is given. All the PBMC samples proliferated in response to 1 ⁇ g/ml of PHA, PPD and SEB.
  • the target cells were autologous PBMC sensitized with 10 ⁇ M of each of the lipopeptides, irradiated and marked with 51 Cr.
  • the chromium release assay was performed after three in vitro stimulations. The cytotoxic activity against autologous EBV cells incubated with the peptides or without peptides was measured in a release assay of 4 hours. The cytotoxic activity was considered as positive when the chromium release was 10% greater than that observed with the target cells alone.
  • A1 and A3 correspond to EBV cells incubated with a group of peptides A1 (n 137-145, n 195-202, n 184-191, n 121-128 for V4.16 or n 183-191, n 121-128 for V4.28) and peptide A3 (n 73-82).
  • the E/T ratio corresponds to 5 ⁇ 10 3 marked target cells, incubated with varying quantities of effector cells.

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DE69834574T2 (de) 2007-05-16
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FR2771640B1 (fr) 2000-02-11
WO1999027954A2 (fr) 1999-06-10
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CA2312043A1 (fr) 1999-06-10
JP2002506001A (ja) 2002-02-26
AU1440099A (en) 1999-06-16

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